LOCUS       sequence-297960-       14962 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral vector for expression of high-specificity eSpCas9(1.1) 
            and sgRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14962)
  AUTHORS   Bauer M, Flatt JW, Seiler D, Cardel B, Emmenlauer M, Boucke K, 
            Suomalainen M, Hemmi S, Greber UF
  TITLE     The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome 
            Release at the Nuclear Pore Complex.
  JOURNAL   Cell Rep. 2019 Dec 17;29(12):3785-3795.e8. doi: 
            10.1016/j.celrep.2019.11.064.
  PUBMED    31851912
REFERENCE   2  (bases 1 to 14962)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2019.11"; journalName: "Cell Rep"; date: 
            "2019-12-17- 17"; volume: "29"; issue: "12"; pages: "3785-3795.e8"
COMMENT     Sequence Label: Lenti-eCas9
FEATURES             Location/Qualifiers
     source          1..14962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2371..2619
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2371..2391
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2542..2561
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4501..4576
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4638..4849
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4864..4873
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4873..4938
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4945..4965
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4990..7527,7528..7530,7531..7992,7993..7995,7996..8163,
                     8164..8166,8167..9090)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9091..9138
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9148..9204
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             9205..9801
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     9698..9718
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9817..10405
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9870..9890)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10288..10299)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10477..10710
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10736..10753)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10742..10966
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11012..11440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11099..11118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11309..11330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11449..11469)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11454..11783
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11634..11769
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11696..11715
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11831..11878
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11897..12271
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    12401..12522
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12438..12457)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12492..12511
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12571..12587)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12571..12587)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12584..12606)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12595..12611
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(12619..12625,12626..12643,12644..12649))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12664..12685
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12802..12819)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12973..13561)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13053..13072)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13732..14523,14524..14592))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     14355..14374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14593..14697)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(14772..14791)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat
     2401 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagacggttg taaatgagca
     2641 cacaaaatac acatgctaaa atattatatt ctatgacctt tataaaatca accaaaatct
     2701 tctttttaat aactttagta tcaataatta gaatttttat gttccttttt gcaaactttt
     2761 aataaaaatg agcaaaataa aaaaacgcta gttttagtaa ctcgcgttgt tttcttcacc
     2821 tttaataata gctactccac cacttgttcc taagcggtca gctcctgctt caatcatttt
     2881 ttgagcatct tcaaatgttc taactccacc agctgcttta actaaagcat tgtctttaac
     2941 aactgacttc attagtttaa catcttcaaa tgttgcacct gattttgaaa atcctgttga
     3001 tgttttaaca aattctaatc cagcttcaac agctatttca caagctttca tgatttcttc
     3061 ttttgttaat aaacaatttt ccataataca tttaacaaca tgtgatccag ctgctttttt
     3121 tacagctttc atgtcttcta aaactaattc ataatttttg tcttttaatg caccaatatt
     3181 taataccata tcaatttctg ttgcaccatc tttaattgct tcagaaactt cgaatgcttt
     3241 tgtagctgtt gtgcatgcac ctagaggaaa acctacaaca tttgttattc ctacatttgt
     3301 gccttttaat aattctttac aatagcttgt tcaatatgaa ttaacacaaa ctgttgcaaa
     3361 atcaaattca attgcttcat cacataattg tttaatttca gctttcgtag catcttgttt
     3421 taataatgtg tgatctatat atttgtttag tttcattttt tctcctatat attcattttt
     3481 aattttaatt ctttaataat ttcgtctact ttaactttag cgttttgaac agattcacca
     3541 acacctataa aataaatttt tagtttaggt tcagttccac ttgggcgaac agcaaatcat
     3601 gacttatctt ctaaataaaa ttttagtaag tcttgtcctg gcatattata cattccatcg
     3661 atgtagtctt caacattaac aactttaagt ccagcaattt gagttaaggg tgttgctctc
     3721 aatgatttca ttaatggttc aatttttaat ttcttttctt ctggtttaaa attcaagttt
     3781 aaagtgaaag tgtaatatgc acccatttct ttaaataaat cttctaaata gtctactaat
     3841 gttttatttt gttttttata aaatcaagca gcctctgcta ttaatataga agcttgtatt
     3901 ccatctttat ctctagctga gtcatcaatt acatatccat aactttcttc ataagcaaaa
     3961 acaaaattta atccgttatc ttcttcttta gcaatttctc tacccattca tttaaatcca
     4021 gttaaagttt ttacaatatt aactccatat ttttcatgag cgattctatc acccaaatca
     4081 cttgttacaa aacttgaata tagagccgga ttttttggaa tgctatttaa gcgttttaga
     4141 tttgataatt ttcaatcaat taaaattggt cctgtttgat ttccatctaa tcttacaaaa
     4201 tgaccatcat gttttattgc cattccaaat ctgtcagcat ctgggtcatt cataataata
     4261 atatctgcat catgtttaat accatattca agcggtattt ttcatgcagg atcaaattct
     4321 ggatttggat ttacaacatt tttaaatgtt tcatcttcaa atgcatgctc ttcaacctca
     4381 ataacgttat atcctgattc acgtaatatt tttggggtaa atttagttcc tgttccatta
     4441 actgcgctaa aaataatttt taaatctttt ttagcttctt gctctttttt gtacgtctct
     4501 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     4561 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg
     4621 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     4681 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     4741 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     4801 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta
     4861 gatgccacca tggactataa ggaccacgac ggagactaca aggatcatga tattgattac
     4921 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
     4981 ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     5041 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     5101 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     5161 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     5221 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     5281 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     5341 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     5401 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     5461 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     5521 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     5581 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     5641 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     5701 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     5761 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     5821 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     5881 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     5941 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     6001 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     6061 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     6121 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     6181 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     6241 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     6301 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     6361 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     6421 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     6481 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     6541 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     6601 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     6661 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     6721 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     6781 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     6841 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     6901 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     6961 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     7021 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     7081 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     7141 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     7201 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     7261 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     7321 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     7381 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     7441 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     7501 gaccatatcg tgcctcagag ctttctggcc gacgactcca tcgacaacaa ggtgctgacc
     7561 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     7621 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     7681 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     7741 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     7801 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     7861 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     7921 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     7981 aaaaagtacc ctgcgctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     8041 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     8101 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     8161 aaggcgcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     8221 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     8281 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     8341 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     8401 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     8461 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     8521 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     8581 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     8641 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     8701 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     8761 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     8821 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     8881 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     8941 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     9001 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     9061 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
     9121 caggcaaaaa agaaaaaggg atccggcgca acaaacttct ctctgctgaa acaagccgga
     9181 gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct cgccacccgc
     9241 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     9301 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     9361 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     9421 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     9481 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     9541 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac
     9601 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     9661 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     9721 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     9781 acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt
     9841 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     9901 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     9961 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
    10021 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
    10081 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
    10141 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
    10201 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
    10261 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
    10321 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
    10381 atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg
    10441 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
    10501 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
    10561 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
    10621 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
    10681 ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg
    10741 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
    10801 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
    10861 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
    10921 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
    10981 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
    11041 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
    11101 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
    11161 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
    11221 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
    11281 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
    11341 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
    11401 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
    11461 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
    11521 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
    11581 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
    11641 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
    11701 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
    11761 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
    11821 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
    11881 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
    11941 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
    12001 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
    12061 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
    12121 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
    12181 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
    12241 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
    12301 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
    12361 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
    12421 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
    12481 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
    12541 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
    12601 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
    12661 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
    12721 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
    12781 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
    12841 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
    12901 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
    12961 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
    13021 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
    13081 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
    13141 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
    13201 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
    13261 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
    13321 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
    13381 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
    13441 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
    13501 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
    13561 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
    13621 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
    13681 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
    13741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
    13801 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
    13861 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
    13921 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
    13981 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
    14041 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
    14101 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
    14161 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
    14221 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
    14281 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
    14341 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
    14401 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
    14461 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
    14521 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
    14581 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
    14641 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
    14701 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
    14761 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
    14821 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
    14881 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
    14941 gtacgggcca gatatacgcg tt
//
